Sialylation of glycoproteins in plants

ABSTRACT

The presence of sialic acids (SAs) at the non-reducing terminal of various glycoconjugates plays important roles in the function and half-life of glycoproteins in mammals and many species of microorganisms. It has been previously unknown that plant cells have the capacity to sialylate glycoproteins. The present invention discloses the presence of sialylated glycoconjugates and identifies N-acetylneuranminic acid (Neu5Ac) and N-glycolylneuraminic (Neu5Gc) on glycoproteins in suspension-cultured cells of plants, for example,  Arabidopsis thaliana  ( A. thaliana ),  Nicotiana tabacum  (tobacco), and  Medicago sativa  (alfalfa), extending the complexity of glycan structures known in the plant kingdom. This invention further discloses methods for engineering plants to produce recombinant glcyoproteins.

CLAIM TO DOMESTIC PRIORITY

This application claims benefit of priority to U.S. provisionalapplication Ser. No. 60/446,477, entitled “Mammalian-Like SialyatedGlycoproteins in Plants”, filed Feb. 11, 2003, by Joshi et al., andwhich is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention concerns generally glycoproteins in animals and plants,and more specifically the sialylated glycoconjugates in plants andmethods for engineering plants to produce recombinant glycoconjugates.

BACKGROUND OF THE INVENTION

Glycosylation is one of the most frequently occurring and importantpost-translational modifications. Most cell surface and secretedproteins are glycosylated in endoplasmic reticulum (ER) and Golgi bycovalent attachment of sugar residues to asparagine (N-glycans) or toserine/threonine (O-glycans) side chains of the proteins (Varki, 1999aand b) (see also, FIG. 1). In vertebrates, sialic acid SA residuesoccupy the non-reducing terminal of most oligosaccharide chains on cellsuface bound and secreted glycoproteins and glycolipids. There areincreasing numbers of reports showing the presence of SAs in bacterialpolysaccharides, Drosophila eggs, insect cell lines, fungi and now plantcells (Schauer, 2000; Joshi et al., 2001; Shah et al., 2003).

In mammals, the non-reducing terminal SA is essential, among otherfunctions, for intermolecular communication and extending the half-lifeof glycoconjugates in circulation. Glycoproteins lacking SA on theirglycan chains are recognized as “foreign” by asialoglycoproteinreceptors and immune cells, removed from the serum and destroyed(Ashwell and Morell, 1974).

SAs are a diverse family of nine-carbon keto-sugar acids (FIG. 2)(Varki, 1992; Yasuo, 1999). More than 40 different species of SAs arefound in nature, and the most common is N-acetylneuraminic acid(Neu5Ac), followed by N-glycolylneuraminic acid (Neu5Gc). Neu5Ac is theprimary SA and most other forms of SAs are metabolically derived from itby hydroxylation (glycosylation), O-acetylation, lactylation,methylation, sulfation or phosphorylation (Varki, 1992). Therefore,according to this disclosure, the terms Neu5Ac and SA are usedinterchangeably. For example, Neu5Gc is synthesized from CMP-Neu5Ac inthe cytoplasm by CMP-Neu5Ac hydroxylase, which is an iron dependentenzyme that utilizes the common electron transport chain of cytochromeb₅ and b₅ reductase (Shaw and Schauer, 1981; Gollub et al., 1998).

The carboxyl group at the 1-carbon position of SA is typically ionizedat physiological pH, giving it a negative charge. SAs can form differentα-linkages from its anomeric carbon to the 3- or 6-position of galactoseresidues or the 6-position of GalNAc residues. The anomeric carbon-2 ofSA also can form linkage to the 8-position of another SA, yieldingpolysialic acid structures as in colominic acids and neural celladhesion molecules (N-CAMs). Combination of all these properties givesstructural and functional diversity to Sas, which can present themselvesin multiple ways on glycoproteins and glycolipids.

In animals, SA metabolism can be divided into three distinct processes:first, synthesis of Neu5Ac in the cytoplasm and its activation toCMP-Neu5Ac in the nucleus; second, transfer of Neu5Ac to the appropriateoligosaccharide acceptor (FIG. 3); and third, removal and degradation ofNeu5Ac, primarily in the lysosome (Reutter et al., 1997).

The de novo synthesis of CMP-Neu5Ac is the result of a complex pathwaythat involves multiple steps in the cytosol beginning with glucose (FIG.3, Keppler et al., 1999). One of the central intermediates in thispathway is UDP-N-acetyl-D-glucosamine (UDP-GlcNAc), which is epimerizedto N-acetyl-D-mannosamine (ManNAc) by UDP-GlcNAc-2-epimerase (Comb andRoseman, 1960). ManNAc is then phosphorylated by ManNAc-6-kinase to formManNAc-6-P. In animals, UDP-GlcNAc-2-epimerase and ManNAc-6-kinaseactivities are found in a single bifunctional enzyme that is highlyconserved and assembles as a hexamer (Hinderlich et al., 1997; Effertzet al., 1999), and the reaction it catalyzes is considered as the firstcommitted step toward SA biosynthesis. UDP-GlcNAc-2-epimerase isallosterically inhibited in a feedback mechanism by CMP-Neu5Ac (Reutteret al., 1997).

Several lines of evidence suggest thatUDP-GlcNAc-2-epimerase/ManNAc-6-kinase catalyze the rate limiting stepsin the formation of SA: a) mutations in the epimerase domain of the GNEgene (Hong and Stanley, 2003) or epigenetically mediated loss of theenzyme expression (Oetke et al., 2003) result in a hyposialylatedphenotype; b) the overall sialylation of cell-surface glycoconjugates ofthe hyposialylated cell lines increases when the medium is supplementedwith ManNAc or D-mannosamine, but not with GlcNAc, D-glucosamine,D-mannose or D-glucose (Keppler et al., 1999).

In animal cells, Neu5Ac-9-phosphate synthase condensesphosphoenolpyruvate (PEP) with ManNAc-6-P to produceN-acetyl-D-neuraminic acid-9-phosphate (Neu5Ac-9-P). Neu5Ac-9-P is thendephosphorylated by Neu5Ac-9-P phosphatase to form Neu5Ac (Lawrence etal., 2000). The Escherichia coli SA synthase gene (neuB), which has beencloned and characterized, encodes an enzyme that directly convertsphosphoenolpyruvate (PEP) and ManNAc to Neu5Ac (Vann et al., 1997). Thusin prokaryotes, the pathway is different in that SA synthase condensesPEP with ManNAc to form Neu5Ac (SA) without any phosphorylation anddephosphorylation steps (Vann et al., 1997; Ringenberg et al., 2003).

SA synthase gene from Drosophila melanogaster and human cDNA librarieshave also been identified based on their high homology to E. coli SAsynthase (neuB) (Kim et al., 2002). It is unclear whetherNeu5Ac-9-phosphate synthase and SA synthase are significantly differentenzymes, therefore, according to this disclosure, the names of these twoenzymes will be used interchangeably.

Neu5Ac made in cytoplasm is then activated by CTP to form nucleotidesugar CMP-Neu5Ac (CMP-SA) in the nucleus; a reaction catalyzed by theCMP-Neu5Ac synthetase (Corfield et al., 1979). Finally, CMP-Neu5Ac istransported to the Golgi apparatus and pumped across its membranes bythe action of a specific antiporter, CMP-Neu5Ac (CMP-SA) transporter.Transport of the nucleotide-sugars to the appropriate compartment of ERor Golgi apparatus is the prerequisite to a successful glycosylationreaction. Nucleotide-sugar transporters are Golgi membrane residenthydrophobic proteins that exist as functional dimers (Gerardy-Schahn etal., 2001).

In animals, CMP-SA transporter facilitates the transport of CMP-SA tothe Golgi lumen in a non-energy dependent fashion. Mammalian cellslacking CMP-SA transporter make incomplete sugar chains, suggesting thatglycosylation may be partially controlled by regulating the transporterand thereby regulating the amount of nucleotide sugar available in theGolgi (Oelmann et al., 2001).

Once CMP-SA is transported into the Golgi lumen, the transfer of SA ontooligosaccharide chains bound to proteins and lipids is facilitated by alarge family of substrate-specific sialyltransferases (STs). Thetransfer of SAs on glycoprotein linked oligosaccharide chains iscatalyzed by STs of alpha-2,3 sialyltransferase (ST3Gal), alpha-2,6sialyltransferase (ST6Gal) and alpha-2,8 sialyltransferase (ST8SA)families that are linkage specific to: α-2,3 (NeuAcα-2,3Galα-1,4GlcNAcor NeuAcα-2,3Galα-1,3GalNAc-O-Ser/Thr), α-2,6 (NeuAcα-2,6Galα-1,4GlcNAcor NeuAcα-2,6GalNAc-O-Ser/Thr), and α-2,8 (NeuAcα-2,8NeuAc) positions,respectively. In animals, the expression of STs is species, organ,tissue and cellular physiology dependent (Taniguchi et al., 2002).

STs are type II membrane proteins with a short NH₂-terminal cytoplasmictail, 16-20 amino acid signal anchor, a highly variable stem region(20-100 amino acids) and a large COOH-terminal catalytic domain that islocalized in the Golgi lumen (Paulson and Colley, 1989). The amino acidsequences of STs show sequence homology in three consensus sequenceregions called sialylmotif L (long), sialylmotif S (short), andsialylmotif VS (very short) (Tsuji et al., 1996). Sialylmotif L isinvolved in the binding of the sugar donor CMP-SA while the sialylmotifS binds to both the donor and the acceptor molecules (Datta et al.,1998). Although the precise function of sialylmotif VS has not beendetermined, it has been suggested that it may be involved in thecatalytic process (Geremia et al., 1997).

In vertebrates, sialic acids (SAs) are the most important andstructurally diverse family of charged sugar residues involved in manybiological and pathological interactions (Schauer, 1985; Kelm andSchauer, 1997). However, it has been as widely accepted that plants donot possess pathways for the biosynthesis of SA and its incorporationinto glycoconjugates (Schauer, 2000).

SUMMARY OF THE INVENTION

The following methods are useful for evaluation of the pathway of SAbiosynthesis and its transfer to the oligosaccharide acceptors andglycoproteins in plants. Any number of plants may be used including, butnot limited to, Arapidopsis thaliana, Nicotiana tabacum (tobacco) andMedicago satiwa (alfalfa).

A method for determining the influence of an enzyme on the sialic acidpathway in a plant is disclosed comprising carrying out an in vitroassay, wherein the assay is capable of determining the enzyme activitiesin an untransformed plant and constitutively over-expressing a gene thatcodes for the enzyme, wherein an effect of over-expression of the geneis capable of being quantified. The enzyme can be mammalian orprokaryotic and may include, but is not limited toUDP-GlcNAc-2-epimerase, ManNAc-6-kinase and sialic acid synthase.

Also disclosed is a method for functionally characterizing a CMP-sialicacid transporter in a plant comprising constitutively over-expressing aplant CMP-sialic acid transporter gene and quantifying an effect ofover-expressing the plant CMP-sialic acid transporter gene. The level ofsialic acid in a plant can be altered be up-regulating ordown-regulating the CMP-sialic acid transporter transcript levels in theplant.

Further disclosed is a method for functionally characterizing asialyltransferase in a plant, comprising constitutively over-expressinga plant sialyltransferase gene and quantifying an effect ofover-expressing the plant sialyltransferase gene. The level of sialicacid in a plant can be altered be up-regulating or down-regulating thesialyltransferase transcript levels in the plant.

Finally, additional features of the disclosed methods are described indetail below and in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the common sugar linkages in mammals;

FIG. 2 shows the parent sialic acid molecule known as Neuraminic acid;

FIG. 3 illustrates sialic acid biosynthesis and transfer toglycoconjugates;

FIGS. 4A and 4B illustrate the specificity of lectin binding tosialylated glycoproteins, specifically biotinylated SNA and MAA bindingto A. thaliana suspension cultured cell total proteins.

FIGS. 5A and 5B show mass spectrometry analysis demonstrating thepresence of Neu5Ac and Neu5Gc on A. thaliana glycoproteins;

FIGS. 6A, 6B and 6C show LC-ESI and MALDI-70F analysis resultsdemonstrating the presence of Neu5Ac (FIG. 6B) and Neu5Gc (FIG. 6C);

FIG. 7 illustrates the topology and amino acid sequence alignment ofselected CMP-SA transporters;

FIG. 8 shows the conserved amino acid motifs and alignment ofsialyltransferase genes from A. thaliana and other organisms;

FIG. 9 is a MALDI MS spectra of standard and plant cell derivedDMB-Neu5Ac (FIG. 9A) and DMB-Neu5Gc (FIG. 9B) illustrating the presenceof Neu5Ac and Neu5Gc on A. thaliana glycoproteins (FIG. 9C) according toan alternate embodiment of the disclosure; and

FIG. 10 is the immunofluorescence microscopic images of plant cells tostudy localization and distribution of sialylated glycocongates withSNA-I: Smbucus nigra agglutinin-I (FIG. 10A), MAA: Maackia amurensisagglutinin (FIG. 10B), and TTM: Tritrichomonas mobilensis lectin (FIG.10C).

DETAILED DESCRIPTION

The present invention discloses plants that produce sialylatedglycoconjugates containing both N-acetyl-D-neuraminic acid (Neu5Ac) andN-glycolyl-D-neuraninic acid (Neu5Gc). Our discovery of sialylatedglycoconjugates in plants establishes a new paradigm in glyobiology,plant biochemistry and proteomics, and opens a new field of research inplant biochemistry and raises important questions regarding thebiosynthesis, regulation, distribution, and function of SAs in plants.The discovery of the presence of SA structures in Arabidopsis thaliana(A. thaliana) and the recognition that biosynthetic pathways arerelatively evolutionarily conserved among animals and bacteria led to anidentification of the key enzymes required for plant SA biosynthesis.

Thus, the following methods are useful for evaluation of the pathway ofSA biosynthesis and its transfer to the oligosaccharide acceptors andglycoproteins in plants. According to one embodiment of the disclosure,the plants are selected from a group comprising Arapidopsis thaliana,Nicotiana tabacum (tobacco) and Medicago satiwa (alfalfa).

Determination of the Influence of MammalianUDP-GlcNAc-2-Epimerase/ManNAc-6-Kinase on the SA Biosynthetic Pathway inPlants.

In mammals, the bifunctional UDP-GlcNAc-2-epimerase/ManNAc-6-kinase is akey enzyme in SA metabolism; It controls the rate-limiting step in SAbiosynthetic pathway and therefore is of importance for the regulation.In bacteria, an enzyme, SA lyase, is coordinately up-regulated with anincrease in SA levels. Enzymes withUDP-GlcNAc-2-epimerase/ManNAc-6-kinase-like functions and significanceexist in A. thaliana. However, extensive homology searches in thedatabase have not identified a candidate A. thalianaUDP-GlcNAc-2-epimerase/ManNAc-6-kinase gene with high sequence homologyto the mammalian or bacterial ones.

Thus, the following method is useful to evaluate the key role ofManNAc-6-P in plant SA biosynthesis: (1) carry out in vitro assays todetermine UDP-GlcNAc-2-epimerase/ManNAc-6-kinase activities inuntransformed plants; (2) constitutively over-express the mammalianUDP-GlcNAc-2-epimerase/ManNAc-6-kinase gene and quantify its impact onSA biosynthesis by measuring free and bound SA; and (3) determine SAlyase activity in transformed and untransformed plants. This method isalso useful in providing insight into the UDP-GlcNAc-2-epimeraseregulation in plant cells.

Determination of the Influence of Mammalian Neu5Ac Phosphate Synthase(SA Synthase) on the SA Biosynthetic Pathway in Plants.

Neu5Ac phosphate synthase (SA synthase) is a cytoplasmic enzyme thatacts downstream of UDP-GlcNAc-2-epimerase/ManNAc kinase in thebiosynthesis of Neu5Ac (SA). In mammals, it catalyzes the synthesis ofNeu5Ac-9-P from ManNAc-6-P and phosphoenolpyruvate (PEP). However, inbacteria, SA synthase condenses ManNAc with PEP to form Neu5Ac.

Because it regulates the flux of ManNAc-6-P/ManNAc entering the SAbiosynthesis and functions in both bacteria and mammals, SA synthase isof importance in SA metabolism. Once again, extensive homology searchesand A. thaliana database have not identified a candidate A. thalianagene with high sequence homology.

Thus, the following method is useful to evaluate the involvement ofNeuAc-9-P as an intermediate in plant SA biosynthesis: (1) carryout invitro assays to determine SA synthase activity in untransformed plants;and (2) constitutively over-express in plants the mammalian SA synthasegene and quantify its effect on plant sialylation by measuring thereaction product (Neu5Ac-9-P or Neu5Ac) and bound SA.

Functionally Characterize A. thaliana CMP-Sialic Acid Transporters.

In animals, CMP-SA transporter facilitates the transport of CMP-SA tothe lumen of the Golgi apparatus. The A. thaliana genome encodes twoproteins with high sequence homology to the mammalian and bacterialCMP-SA transporters. The following method is useful for determining thatplant orthologs function to facilitate the transport of CMP-SA to thelumen of the Golgi apparatus: (1) constitutively over-express in plantsthe putative CMP-SA transporter genes; (2) down-regulate endogenousCMP-SA transporter transcript levels in plants using techniques such aspost-transcriptional gene silencing (RNAi); (3) characterize CMP-SAtransporter T-DNA mutants of; and (4) determine the levels ofsialylation in untransformed, CMP-SA transporter-up-regulated, andCMP-SA transporter-down-regulated plants using molecular and biochemicalapproaches.

Functionally Characterize A. thaliana Sialyltransferases (STs).

In mammals, sialyltransferases (STs) transfer SA residues from CMP-SA tothe terminal position on asialo-oligosaccharides bound to proteins andlipids. Homology searches in the A. thaliana genome database haveidentified three candidate genes for plant STs. Therefore, the followingmethod is useful for determining that the sequence orthologs ofmammalian STs in the A. thaliana genome encode enzymes that catalyzesimilar reactions: (1) constitutively over-express selected putative STgenes in the plants; (2) down-regulate endogenous ST transcript levelsin plants using techniques such as post-transcriptional gene silencing(RNAi); (3) characterize ST T-DNA mutants of the plants; (4) determinethe levels of sialylation of endogenous total proteins in untransformed,ST-up-regulated, and ST-down-regulated plants using molecular andbiochemical approaches; and (5) express plant STs in E. coli and performbiochemical characterization.

Experimental Method for Determining Presence of SA.

1. Detection of Sialylated Glycoproteins in A. thaliana Cells by LectinBinding.

Glycoproteins from A. thaliana suspension-cultured cells grown oninorganic salts and sucrose were probed with a mixture of biotinylatedSNA (Sambucus nigra) and MAA (Maackia amurensis) lectins, revealingterminal SAa-2,6-Gal and SAα-2,3-Gal structures (Shibuya et al., 1987;Wang and Cummings, 1988). As illustrated in FIG. 4, specificity oflectin binding to sialylated glycoproteins was verified by using fetuin,asialofetuin and inhibition of lectin binding by 100 mM lactose. FIG. 4shows biotinylated SNA and MAA binding to A. thaliana suspensioncultured cell total proteins. Positions of molecular weight standardsare indicated to the left.

In FIG. 4A, total cell proteins from A. thaliana were resolved on 4-20%SDS/PAGE and transferred on PVDF membranes. Sialylated glycoproteinswere detected colorimetrically (Sigmafast NBT/BCIP tablets) by bindingof biotinylated SNA and MAA and avidin-alkaline phosphatase (Vectorlabs, CA) probes with 100 mM lactose, which inhibited binding of lectinsto A. thaliana glycoproteins.

To further confirm these results, in FIG. 4B, sialylated glycoproteinswere affinity-purified through immobilized SNA and MAA columns, digestedwith α-2-3,6 sialidase from Clostridium perfringens and probed with SNAand MAA lectins. The affinity-purified and sialidase-digested proteinsdid not bind to the lectins, confirming removal of α-2-3,6 linked SAsfrom A. thaliana glycoproteins.

2. Purification and Separation of SA by RP-HPLC and Mass Spectrometry.

SAs from A. thaliana proteins were released by mild acid hydrolysis (2 Macetic acid, 80° C. for 3 hr), purified through ion exchangechromatography and labeled with the fluorescent dye1,2-diamino-4,5-methylene dioxybenzene (DMB, λ_(ex) 373 nm, λ_(em) 448nm). DMB-SA derivatives were separated on a reverse-phase C18 column onan Agilent 1100 workstation under isocratic conditions of 9%acetonitrile, 7% methanol and 84% water at flow rate of 0.65 ml/min(Hara et al., 1987).

In FIG. 5A, commercially available Neu5Ac and Neu5Gc were used asstandard SAs. As shown by FIG. 5B, in A. thaliana samples, a prominentpeak corresponding to Neu5Gc and a smaller peak corresponding to Neu5Acwere detected, indicating the presence of these two SAs on A. thalianaglycoconjugates. Similar results were observed when SAs were removedfrom glycoconjugates by α-2-3,6 sialidase treatment.

In FIG. 6, fractions collected from HPLC were concentrated and subjectedto analysis using LC-ESI and MALDI-TOF. The DMB-labeled standards areshown in FIG. 6A. DMB-Neu5Ac m/z at 426 (FIG. 6B) and DMB-NeuGc m/z at442 (FIG. 6B) confirm the presence of Neu5Ac and Neu5Gc respectively.

3. Detection of Sialylated Glycoconjugates by ImmunofluorescenceMicroscopy as Shown in FIG. 10.

A. thaliana suspension-cultured cells (top panel) were fixed in 4%formaldehyde and probed with biotinylated lectins (SNA-I, MAA, TTM),specific for sialic acid to study cell surface expression of sialicacid. Secondary probe was anti-biotin antibody conjugated to Cy2 (λ_(ex)495 nm, λ_(em) 519 nm). To study intracellular distribution ofsialoglycoconjgates, protoplasts (bottom panel) were prepared bydigesting formaldehyde-fixed cells with cellulase and pectinase enzymesat 30° C. for 3 hours. Protoplasts were treated with 10% triton X-100and probed with lectins as mentioned earlier. For all the experiments,ConA-Alexa568 (λ_(ex) 579 nm, λ_(em) 603 nm) was used as a positivecontrol and 1% BSA was used to inhibit non-specific binding. Sialicacid-specific lectins did not show binding to the cell surface. However,binding of these lectins was significant when protoplasts were prepared.

Sialylated glycoconjugates in suspension-cultured cells were detectedusing biotinylated SNA and MAA lectins. Anti-biotin antibodiesconjugated to Cy2 were used to detect sialylated glycoconjugates usingconfocal microscopy. Cells were treated with pectinase and cellulase todetect intracellular sialoglycoconjugates or this treatment was omittedto detect cell-surface expression of sialoglycoconjugates. Plant cellswere treated with BSA to prevent nonspecific binding and observed with aconfocal microscope. These experiments suggest that SA motifs areprimarily located on intracellular and/or cell membrane glycoconjugates,but not on the plant cell wall, as shown by the comparison of cells andprotoplasts for TTM (FIG. 10A), SNA (FIG. 10B) and MAA (FIG. 10C).

4. Organ-Specific Distribution of Sialylated Glycoproteins in A.thaliana Plants.

A. thaliana plants were grown in a 16/8 light cycle at 23° C. Aftermaturation, different organs of A. thaliana were collected and totalprotein extracts from stem, rosette leaves, reproductive leaves andflowers were prepared. Proteins were separated on SDS-PAGE and probedwith SNA-I and MAA to study the distribution of sialylatedglycoconjugates in different organs.

The preliminary results show that sialoglycoproteins are differentiallydistributed in all organs of the A. thaliana plant. Quantification ofSAs by the ferri-corcinol method suggested approximately 2% sialylationof A. thaliana total proteins. Sialylation was also detected in culturedcells of other plant species, such as Nicotiana tabacum and Medicagosativa.

5. Data Mining.

Nucleotide and protein sequences encoding experimentally-authenticatedUDP-GlcNAc-2-epimerase/ManNAc-6-kinases, SA synthases, CMP-SAtransporters, and STs from Homo sapiens, Mus musculus, Rattusnorvegicus, Drosophila melanogaster, and Sus scrofa were queried againstA. thaliana database using BLASTN and BLASTP algorithms. In the case ofUDP-GlcNAc-2-epimerase/ManNAc-6-kinase and SA synthase, no A. thalianagenes were identified that showed significant sequence homology (anexpect value <1E-4). However, queries using CMP-SA transporters and STs(α-2,3-, α-2,6-, and α-2,8-) from each of the aforementioned organismsidentified A. thaliana genes encoding proteins with significant homology(>1E-4). Two possible candidates (AT5G41760 and AT3G59360) for CMP-SAtransporters were found: AT5G41760 shows the highest degree of homologyto the Mus musculus CMP-SA transporter (1E-27), while AT3G59360 ishomologous (1E-17) to the Homo sapiens CMP-SA transporter. Both proteinsare predicted to have 10 membrane spanning regions based on Kyte andDoolittle hydrophobicity algorithms.

FIG. 7 shows the topology and amino acid sequence alignment of selectedCMP-SA transporters. FIG. 7 shows an alignment of these A. thalianacandidates with other CMP-SA transporters in regions previouslyidentified by Olemann et al. (2001) to be critical for CMP-SAtransporter function. Also shown is a diagram generated by Eckhardt etal. (1999) illustrating experimentally-determined murine CMP-SAtransporter topology that we have modified to show a relationship withthe alignment below it.

Modifications in the Mus musculus CMP-SA transporter of Sα-213 and G281Dameliorated transporter activity while Y122H did not (Olemann et al.,2001). Surprisingly, both A. thaliana candidate genes have conservedtyrosine residues at a position correlating to Y122 in the mouse CMP-SAtransporter, but only AT5G41760 has a conserved serine corresponding tothe mouse S213. Neither candidate A. thaliana gene has the conservedglycine at position 281. The relevance of these residues as they relateto CMP-SA transporter function in A. thaliana can ultimately bedetermined experimentally.

BLAST analysis of STs from the organisms listed above identified threehomologs in A. thaliana. AT1G08280 shows the highest degree of homologyto mouse α-2,3 type 4A ST (2E-12), while the other two A. thalianacandidates AT3G48820 and AT1g08660 show greatest homologies to humanα-2,3 type 4B ST (6E-14) and human α-2,8 ST, respectively (5E-11).

Depicted in FIG. 8 is a diagram of a typical ST with salient featuresnoted. In most STs, the transmembrane domain and the three sialylmotifs,L, S and VS are conserved. FIG. 8 also shows an alignment of residues insialylmotif L of mammalian and A. thaliana genes. The three putative STsfrom A. thaliana are highly conserved in the sialylmotif L.

Supplemental Methods for Determining Presence of SA.

1. Preparation of Cell Extract.

All experiments were carried out with 7-day old suspension culturedcells of Arabidopsis thaliana. Cells were washed with water andsuspended in resuspension buffer (25 mM HEPES pH 7.5, 2 mM DTT, 0.5 mMPMSF and 1 μg/ml leupeptin). Total protein extract was prepared bysubjecting to French-press (American Instrument Company, MO) at 10,000psi. Membrane-bound proteins were extracted with 0.05% β-dodecylmaltoside. Samples were centrifuged at 5000 rpm for 20 minutes at 4° C.,supernatant was collected and passed through PD10 columns (Amersham, NJ)for removal of salts and detergent. Eluant from PD10 column was used astotal cell protein extract.

The total amount of protein was determined by Bradford proteinquantification method (Bio-rad, CA) according to supplier'sinstructions. For sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) analysis, 15 μg A. thaliana proteins wereused (FIG. 1). 0.5 μg fetuin and asialofetuin were loaded on theSDS-PAGE as positive and negative controls, respectively (fetuin andasialofetuin can be detected at 100 ng levels by sialic acid specificlectins used for the analysis).

Lectin Blot and Lectin-Affinity Chromatography.

Proteins were separated on SDS-PAGE and electro-transferred ontoPolyvinylidene fluoride (PVDF) membrane. Membranes were washed threetimes (10 min.×3) in Tris-Buffered Saline (TBS) (50 mM Tris-HCl, pH 7.5,150 mM NaCl) and blocked in 1% gelatin containing 0.05% Tween 20™ for 3hours at room temperature with gentle shaking. Membranes were thenincubated in lectin incubation buffer (50 mM Tris-HCl, pH 7.5, 150 mMNaCl, 1 mM CaCl₂, 1 mM MgCl₂, 1 mM MnCl₂) containing biotinylated SNA-I(10 μg) and MAA (50 μg) lectins for 2 hr. Membranes were washed threetimes with TBS (10 min.×3) and incubated in streptavidin-conjugatedalkaline phosphatase for 1 hour.

Membranes were washed three times (10 min.×3) and lectin binding tospecific glycan residues was calorimetrically detected by5-Bromo-4-chloro-3-indolyl phosphate/Nitro Blue Tetrazolium (Sigmafast™BCIP/NBT, Sigma, MO). For lactose inhibition experiment, lectins werepre-incubated in 100 mM lactose solution. For this experiment, blockingbuffer, lectin incubation buffer as well as the wash buffer contained100 mM lactose.

Sepharose-conjugated SNA-I and MAA columns were equilibrated at roomtemperature with TBS (10 mM tris-HCl, pH 7.5, 150 mM NaCl, 1 mM CaCl₂, 1mM MgCl₂, 1 mM MnCl₂). Total protein extract from A. thaliana was loadedon the lectin-affinity column, washed with TBS (at least five times thecolumn volume) to remove unbound proteins. Sialic acid containingproteins were eluted using 100 mM lactose in TBS and 20 mM unbufferedethylenediamine.

The column eluant was dialyzed and concentrated using Macrosepcentrifugal devices (VWR, PA). The eluant was also subjected todigestion with α2-3,6 sialidase (Clostridium perfringens, Calbiochem,CA). Affinity-purified proteins that were sialidase treated anduntreated, were separated on SDS-PAGE, transferred on to PVDF membraneand detected with SNA-I and MAA lectins.

Purification of Sialic Acids from A. thaliana Cells.

A. thaliana cell extract was prepared as described above and proteinswere precipitated using ammonium sulfate (72%) to eliminate thepossibility of contamination with KDO. Precipitated proteins weredialyzed against water overnight at 4° C. by change of water at leastthree times using spectrapore dialysis membrane (MWCO: 2000 Da, Pierce,Ill.) and then used for release of sialic acids. Sialic acid residueswere released from dialyzed proteins by subjecting to mild acidhydrolysis (2M acetic acid, 80° C., 3 hour) in presence of 1% ButylatedHydroxytoluene (BHT). Acid-hydrolyzed protein samples were incubated onice for 30 minutes.

Released sialic acids were collected by dialyzing overnight against H₂Ousing spectrapore dialysis membrane (MWCO: 1000 Da, Pierce, Ill.) at 4°C. Diffusate from the membrane was collected, lyophilized andsubsequently resuspended in water. Sialic acid samples were loaded onDowex AG-50WX2 (Hydrogen) column (Pierce, Ill.) and washed with water.Eluant was collected and pH was adjusted with 10 mM sodium formate pH5.5 buffer. Eluant was then subjected to Dowex AG50×8 (formate) column(Bio-Rad, CA). The column was washed using 10 mM formic acid. Sialicacids were eluted using 1 M formic acid. Eluant was lyophilized,reconstituted in water, labeled with 1,2-diamino-4,5-methyleneoxybenzene(DMB, a fluorescent dye specific for sialic acids) and analyzed onRP-HPLC (as shown in FIG. 2) or mass spectrometry.

Structural Analysis of Sialic Acids.

DMB-SA derivatives were separated on a reversed-phase C18 column(Phenomenex, CA) on an Agilent 1100 workstation under isocraticconditions of 9% acetonitrile, 7% methanol and 84% water at flow rate of0.65 ml/min. As shown in FIG. 4A, commercially available Neu5Ac andNeu5Gc were used as standard SAs. In A. thaliana samples, a prominentpeak corresponding to Neu5Gc and a smaller peak corresponding to Neu5Acwere detected, indicating the presence of these two SAs on A. thalianaglycoconjugates.

Results similar to FIG. 4 were observed when sialic acids were removedfrom glycoconjugates by α2-3,6 sialidase treatment. HPLC-purified DMB-SAderivatives were used for further analysis, using MALDI-TOF (VoyagerDE-STR, Applied Biosystems). As shown in FIG. 9, the parent ion massesof sialic acids purified from A. thaliana were confirmed by comparingthem with commercially available standard Neu5Gc and Neu5Ac (FIG. 9A).The m/z at 426 confirms the presence of Neu5Ac (FIG. 9B) and m/z at 442confirms the presence of Neu5Gc (FIG. 9C).

Methods for Engineering Plant Pathways

These genomic and experimental data clearly show that plants containgenes involved in sialylation and produce sialylated glycoconjugates.However, none of the biosynthetic and regulatory steps of the SAbiosynthesis pathway are known in plants. This detailed descriptionfurther discloses specific metabolic engineering approaches that willassist in the understanding and establishment of the molecular andbiochemical mechanisms of SA biosynthesis pathway in plants.

The presence of sialylated glycoproteins in A. thaliana raises importantquestions about SA synthesis, transport and transfer, for which thescientific community still has no experimental knowledge. Fortunately,there are extensive studies on SA biosynthesis in animals and bacteriathat clearly show that these organisms utilize similar metabolicpathways and that the enzymes that catalyze identical chemical reactionsshare relatively high homology on amino acid level. This evolutionaryconservatism defines the identification of genes/proteins by comparativegenomic/proteomic database analyses as the first logical step inelucidating the biosynthesis of SA in plant cells.

As described in our preliminary studies, in animals,UDP-GlcNAc-2-epimerase/ManNAc-6-kinase and NeuAc phosphate synthase playcrucial roles in SA synthesis. The epimerase domain of the mammalianUDP-GlcNAc-2-epimerase/ManNAc-6-kinase shares high level of sequencehomology to bacterial UDP-GlcNAc-2 epimerase, as do the bacterial SAsynthase and mammalian NeuAc phosphate synthase genes.

The absence of plant homologs to mammalianUDP-GlcNAc-2-epimerase/ManNAc-6-kinase and NeuAc phosphate synthase(bacterial SA synthase), requires further examination of whether theseenzyme activities exist in the plant cells, as the possibility thatdifferent protein entities might have the same or similar functionalcharacteristics could not be ruled out.

Overexpression of human UDP-GlcNAc-2-epimerase/ManNAc-6-kinase and NeuAcphosphate synthase genes in A. thaliana followed by measurement of theamount of SA and levels of sialylation compared to untransformed plants,would support or overrule the involvement of these enzymes and theirreaction products in plant SA biosynthesis pathway. The human genesencoding UDP-GlcNAc-2-epimerase/ManNAc-6-kinase and NeuAc phosphatesynthase will be received from our collaborators, Prof. Werner Reutter(Freie University, Berlin, Germany) and Prof. Michael Betenbaugh (JohnsHopkins University, MD), respectively.

Transport of CMP-SA to Golgi lumen and the subsequent transfer of SAs tothe non-reducing termini of the oligosaccharide chains ofglycoconjugates are conserved processes among animals, insects andplants. Several putative CMP-SA transporter and ST genes have beenidentified in A. thaliana database by using their mammalian andDrososphila counterparts as queries. Plant CMP-SA transporters and STscan be functionally characterized by transgenic approach, using both,gene over-expression and down-regulation.

The results from the impact of these manipulations on the amount of SAand sialylation levels provide an evaluation of gene product function,and enable identification of A. thaliana sialylated glycoproteins,further elucidating the biological significance of their terminal SAresidues. For down-regulation experiments, collections of targeted andrandom T-DNA mutants from various sources can be used.

We have identified multiple T-DNA mutants for each of the A. thalianagenes described above (in many cases multiple T-DNA insertions atdifferent gene positions are available for the same gene). The presenceand location of the T-DNA can be verified by PCR or hybridizationtechniques, and the plants can then be analyzed for alterations in thelevels of sialylated proteins and free SA.

Because functional redundancy may, at times, result in the effect ofT-DNA insertional inactivation being somewhat unclear, theimplementation of RNAi technology is used as a complementary approach.A. thaliana STs will also be overexpressed in E. coli, to ensure quickproduction of enough amount of functionally active proteins that will beused for further biochemical characterization.

The experimental protocols and assays described below are non-limitingexamples for practicing the methods described herein for differenttissues of A. thaliana plants.

Vector Construction for Plant Transformation.

1. Construction of Plant Binary Vectors for Agrobacterium-MediatedTransformation.

A GATEWAY cloning technology (Invitrogen) that exploits site-specificrecombination system of bacteriophage λ of E. coli can be used forconstruction of plant expression vectors. The cDNAs—fragments (for hpRNA constructs) or whole length, can be PCR-amplified (Pfu Turbo DNApolymerase, Stratagene), in the latter case with (because of the lack ofantibodies specific for sialyltransferases, CMP synthetase, CMPtransporter and UDP-GlcNAc-2-epimerase/ManNAc-6-kinase), or without amyc epitope appended to the N-terminus. A directional Topo®-clonningstrategy is implemented to position them between the attL sites ofpENTR/SD/D-Topo vector (Invitrogen).

By the means of LR reaction (Invitrogen), the target genes can betransferred to GATEWAY-compatible binary vectors under the control ofCaMV ³⁵S promoter and nos terminator. A pKZ7WG2 and pK7GWIWG2 is usedfor over-expression and co-suppression of the target genes,respectively. Both vectors are built within the pPZP200 backbone andcontain streptomycin/spectinomycin resistance gene for bacterialselection and kanamycin resistance plant selectable marker gene (Karimiet al., 2002). Between the inverted GATEWAY fragments pK7GWIWG2 containsArabidopsis intron (ac007123.em_pl) with ideal features for efficientsplicing (A+T-rich with a branch site close to the consensus). Inplants, it produces double-stranded RNA (hairpin RNA) from the insertedsequence of interest, thus triggering in an efficient waypost-transcriptional gene silencing.

2. Construction of Expression Vectors for E. coli Transformation.

The cDNAs can be amplified by PCR (Pfu Turbo DNA polymerase, Stratagene)with appropriate linker sequences (if necessary) and cloned into themultiple cloning site of pET14b (Novagen) by standard restrictiondigestion and ligation procedures. pET14b is a bacterial expressionvector employing a T7 promoter/terminator and an in-frame N-terminalpoly histidine (6×His) affinity tag that yields high levels ofexpression in E. coli after IPTG induction and allows easy detection andpurification of the recombinant protein.

Transformation of Arabidopsis thaliana.

1. Agrobacterium-Mediated Transformation of Arabidopsis Plants.

Stable Agrobacterium-mediated transformation in planta is carried outusing vacuum-infiltration procedure as described by Clough and Bent(1998). After 24 hours co-cultivation, the plants are rinsed with waterand grown to maturity. Seeds are collected, surface sterilized and grownon selection medium (0.5×MSO containing 0.7% (w/v) agar, supplementedwith 25-50 microgram/ml kanamycin). The transformants are transferred tosoil and their transgenic status confirmed by molecular analyses.

2. Biolistic Transformation of Suspension Culture Cells (for SubcellularLocalization).

Cells from four day post-subcultured Arabidopsis suspension cultures arecollected by centrifugation, resuspended in an equal volume oftransformation medium-TM (growth medium without kinetin and NAA,supplemented with 250 mM sorbitol and 250 mM mannitol), spread on filterpapers and allowed to equilibrate for 1 hours at RT. A microprojectilebombardment (PDS 1000/He Bio-Rad) with plasmid DNA precipitated ontoM-17 tungsten particles will be carried out as described by Banjoko andTrelease (1995). Cells are left in darkness for 2, 5, 10, or 20 hours toundergo post-bombardment transient gene expression before the analysis.

3. Immunofluorescence Microscopy.

Bombarded cells are fixed in 4% (w/v) formaldehyde in 0.5×Arabidopsis TMfor 1 h at RT, washed three times in phosphate buffered saline (PBS, 4.3mM Na₂HPO₄, 1.4 mM KH₂PO₄, 2.7 mM KCl, 137 mM NaCl, pH 7.4) andincubated in 0.1% (w/v) Pectolyase Y-23 and 0.1% (w/v) Cellulase-RS(Karlan Research Products, Santa Rosa, CA) for 2 hours at 30° C. Duringincubation plasma and organell membranes are permeabilized by treatmentwith 0.3% (v/v) Triton X-100 for 15 min at RT. Fixed and permeabilizedcells are processed for indirect immunofluorescence as describedpreviously (Flynn et al., 1998). Primary and dye-conjugated secondaryantibodies are diluted as necessary.

4. DNA and RNA Analyses.

Isolation of plasmid and genomic DNA, RNA isolation, Southern andNorthern blot analyses, PCR and RT-PCR are carried out according tostandard protocols (Ausubel et al., 2001).

5. SDS PAGE, Western and Lectin Blot Analyses.

Proteins are prepared and separated on 4-20% Ready Gel® Precastpolyacrylamide gels (Bio-Rad). For Western analyses, proteins aretransferred to PVDF membranes in a semi-dry transfer apparatus (Bio-Rad)employing an anode (0.3 M Tris, 0.1 M glycine, 0.0375% SDS)/cathode (0.3M aminocaproic acid, 0.03 M Tris, 0.0375% SDS) buffering system. Theblots are blocked with 5% (w/v) instant milk (Carnation Brand) inTris-buffered saline for 1 hour.

The target proteins are detected using specific primary antibodies andalkaline phosphatase-coupled secondary antibodies diluted (1:10,000) inTBST containing 1% (w/v) dried milk. Immunoblots are washed three timesfor 10 min with TBST before and after antibody incubations. Lectinanalyses are carried out by Roche Molecular Biochemicals protocol. Theblots are imaged using Immunostar substrate (Bio-Rad) andautoradiography film.

6. Purification of Recombinant Proteins Expressed in E. coli.

A fermentor-based-cell production of the target proteins, expressed in Ecoli, is performed as described by Panitch et al. (1997). Preparation ofthe protein samples and purification of His-tagged proteins of interestby metal chelation affinity chromatography follow the Novagen protocols.If the recombinant proteins require additional purification, additionalchromatography methods are employed.

7. Determination of Free and Bound Sialic Acids.

Colorimetric assays adapted to the microscale and HPAEC (High pH AnionExchange Chromatographic) are used to detect and quantify SAs. Bound SAsare the estimated by the periodate-resorcinol microtiter plate assaycapable of detecting nano mole amounts (Bhavanandan and Sheykhnazari,1993). The advantage of this approach is that there is no need forpretreatment and even crude samples can be analyzed. Free SAs areestimated by the micro scale version of the Warren thiobarbituric acidmethod (Yeh et al., 1971).

This widely used colorimetric assay was believed to be specific for freeSA; however, recently it was shown that the reagent also reacts with SAlinked 2, 6 to unsubstituted GalNAc (Bhavanandan et al., 1998). Further,compounds that are present in plants such as KDO(3-deoxy-D-manno-2-octulosonic acid) and KDN(2-keto-3-deoxy-D-glycero-D-galacto-nonic acid) are also known to givepositive signals in the periodate-thiobarbituric acid assay (Kitajima etal., 1992). Therefore, it is essential to confirm that a positivereaction in this assay is indeed due to free SAs by additionaltechniques.

Thus, all samples are also analyzed by HPAEC. Using this technique wewere able to detect pico mole quantities of SA and also distinguishesbetween various forms of SA. Samples containing bound SA are firsttreated with mild acid (0.1 N sulfuric acid, 80 to 60 min) to hydrolyzethe ketosidic bond. The samples containing free SA and the hydrolysatesare analyzed on a CarbPac PA-10 column (Dionex Corp.) and using pulsedamperometric detection (Manzi et al., 1990; Rohrer, 2000).

If any unusual SA forms are detected by HPAEC in the plant samples,these are isolated by RP-HPLC, derivatized with DMB and analyzed byLC-ESI and MALDI-TOF mass spectrometry as described above.

8. Enzymatic Assays.

The following enzymatic assays are then carried out with the appropriatemodifications required for plant sample analyses.

A. UDP-GlcNAc 2-Epimerase and ManNAc-6-Kinase.

UDP-GlcNAc 2-epimerase and ManNAc-6-kinase activity is measured by amethod described by Effertz et al (1999). In brief, known quantities oftotal soluble proteins from the plant cell extracts are incubated withthe reaction mixtures for UDP-15. GlcNAc 2-epimerase andManNAc-6-kinase. For UDP-GlcNAc-2-epimerase assay, the mixture includes:45 mM Na₂HPO₄, pH 7.5, 10 mM MgCl₂, 1 mM UDP-GlcNAc, 25 nCi ofUDP-[14C]GlcNAc. For the ManNAc kinase assay the mixture contains: 60 mMTris/HCl, pH 8.1, 20 mM MgCl₂, 5 mM ManNAc, 50 nCi of [14C]ManNAc, 20 mMATP (disodium salt).

After incubation for pre-determined time, the reaction is stopped by theaddition of ice-cold ethanol. Radiolabeled products are separated bypaper chromatography, eluted from the paper and estimated by liquidscintillation counting. One unit of enzyme activity is defined as thatcatalyzing the formation of 1 mmol of product/min at 37° C. Specificactivity is expressed as milliunits/mg of protein.

B. SA Synthase.

In vitro activity assay is carried out according to Lawrence et al(2000). 5 μl of substrate solution is incubated with 20 μl of celllysate (30 or 60 min) at 37° C. The substrate solution contains 10 mMMnCl₂, 20 mM PEP, and either 5 mM ManNAc, 5 mM ManNAc-6-P or 25 mMmannose 6-phosphate. Assays performed on samples containing boiledlysates can be used as controls.

Following incubation, all samples are boiled for 3 min, centrifuged for10 min at 12,000×g, and split into two 10-ml aliquots. One aliquot istreated with 9 units of calf intestine alkaline phosphatase (RocheMolecular Biochemicals) along with 3 ml of accompanying buffer, whilethe other aliquot is diluted with water and buffer. Alkaline phosphatase(AP)-treated aliquots are incubated for 4 hours at 37° C., and 10 μl ofboth AP-treated and -untreated samples are reacted with DMB as describedabove.

C. CMP-Sialic Acid Transporter.

The in vitro transport assay is performed essentially as describedpreviously (Aoki et al., 2001) with slight modifications. The reactionis started by addition of the plant cell membrane preparation to thereaction mixture (0.8 M sorbitol, 10 mM Tris-HCl (pH 7.0), 1 mM MgCl₂,0.5 mM dimercaptopropanol, and ³H-labeled nucleotide sugar-CMP-3H SA.The reaction is stopped using ice-cold stop buffer (0.8 M sorbitol, 10mM Tris-HCl (pH 7.0), and 1 mM MgCl₂), and poured onto a nitrocellulosefilter. The filter is washed three times with stop buffer and dried.After solubilization, the radioactive material on the filters isestimated liquid scintillation counting.

D. Sialyltransferase (ST).

ST activity is assayed according to Carey and Hirschberg (1980). Inbrief, membrane fraction of the plant cells prepared by sucrose stepgradient centrifugation is incubated with the reaction mixture. Atypical reaction mixture contains CMP-3H-sialic acid [0.3 mCi],asialofetuin (and other suitable acceptors), and buffer [33 mM Na₃PO₄,100 mM NaCl, pH 7.5, and 0.2% Triton X-100]. Controls include reactionmixtures minus cell fractions or asialofetuin. Fetuin has both N-linkedand O-linked saccharides and therefore, asialofetuin is a good initialsubstrate to measure total activity of sialyltransferase(S).

Subsequently, specific asialosubstrates, such those derived fromalpha-1-acid glycoproteins or mucins, are tested to distinguish thesubstrate specificity of plant sialyltransferases. The reaction isstopped by addition of cold 1% phosphotungstic acid (PTA) in 0.5 N HCl.The precipitated glycoproteins are pelleted by centrifugation and rinsedthree times with fresh 1% PTA/0.5 N HCl. The pellet is dissolved in 1 NNaOH transferred to a scintillation vial and neutralized with 4 N HCl.The product radioactivity is estimated by liquid scintillation counting.

E. SALyase.

SA lyase is partially purified from extracts of A. thaliana by ammoniumsulfate precipitation method. Activity of lyase is measured by addingknown amount of exogenous SA at 37° C. for 15 minutes. The reaction isstopped by heating the reaction mixture at 100° C. Production ofpyruvate (degradation product of SA) is measured by adding lactatedehydrogenase and NADH. As pyruvate is converted to lactate, NADH isoxidized to NAD. Oxidation of NADH (production of pyruvate) can beestimated by measuring the change of the absorbance at 340 nm. One unitof enzyme is defined as the quantity that yields 1.0 μmole of pyruvatein above-described conditions.

Various embodiments of the invention are described above in the Drawingsand Description of Various Embodiments. While these descriptionsdirectly describe the above embodiments, it is understood that thoseskilled in the art may conceive modifications and/or variations to thespecific embodiments shown and described herein. Any such modificationsor variations that fall within the purview of this description areintended to be included therein as well. Unless specifically noted, itis the intention of the inventor that the words and phrases in thespecification and claims be given the ordinary and accustomed meaningsto those of ordinary skill in the applicable art(s). The foregoingdescription of a preferred embodiment and best mode of the inventionknown to the applicant at the time of filing the application has beenpresented and is intended for the purposes of illustration anddescription. It is not intended to be exhaustive or to limit theinvention to the precise form disclosed, and many modifications andvariations are possible in the light of the above teachings. Theembodiment was chosen and described in order to best explain theprinciples of the invention and its practical application and to enableothers skilled in the art to best utilize the invention in variousembodiments and with various modifications as are suited to theparticular use contemplated. Therefore, it is intended that theinvention not be limited to the particular embodiments disclosed forcarrying out this invention, but that the invention will include allembodiments falling within the scope of the appended claims

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1-47. (canceled)
 48. A method for producing recombinant sialylatedglycoproteins in plants, comprising administering a vector comprising anucleic acid encoding the glycoprotein to a plant cell, wherein theplant cell expresses a plant CMP-sialic acid transporter and a plantsialyltransferase.
 49. The method of claim 48, wherein the plant cell isa Arabidopsis thaliana, Nicotiana tabacum, or Medicago stativa plantcell.
 50. The method of claim 48, wherein the plant cell comprises anexpression vector encoding plant CMP-sialic acid transporter or plantsialyltransferase.
 51. The method of claim 48, wherein the CMP-sialicacid transporter is encoded by a nucleic acid comprising a genedesignated as AT5G41760 or AT3G59360.
 52. The method of claim 48,wherein the plant sialyltransferase is encoded by a nucleic acidcomprising a gene designated as AT1G08280, AT3G48820, or AT1g08660 gene.53. A method for engineering plants to produce recombinant sialylatedglycoproteins, comprising administering to the plant cell a vectorcomprising a nucleic acid encoding plant CMP-sialic acid transporter andplant sialyltransferase.
 54. The method of claim 53, wherein the plantcell is a Arabidopsis thaliana, Nicotiana tabacum, or Medicago stativaplant cell.
 55. The method of claim 53, wherein the CMP-sialic acidtransporter is encoded by a nucleic acid comprising a gene designated asAT5G41760 or AT3G59360.
 56. The method of claim 53, wherein the plantsialyltransferase is encoded by a nucleic acid comprising a genedesignated as AT1G08280, AT3G48820, or AT1g08660 gene.
 57. The method ofclaim 53, further comprising administering to the plant cell a vectorcomprising a nucleic acid encoding human UDP-GlcNAc-2-epimerase,ManNAc-6-kinase and NeuAC phosphate synthase.